Advances in recombinant DNA technology have made possible the generation of a variety of vaccines, such as subunit vaccines and DNA-based vaccines. These are in addition to the more traditional killed or attenuated vaccines. Adjuvants that enhance the immune system's response to antigenic material are known; however, currently, few adjuvants are approved for human usage, although additional adjuvants are in pre-clinical and clinical studies.
The ADP-ribosylating bacterial toxins, a group of potent toxins to humans, include diphtheria toxin, pertussis toxin (PT), cholera toxin (CT), the E. coli heat-labile toxins (LT1 and LT2), Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins, as well as toxins from C. perfringens, C. spiriforma and C. difficile. These toxins are composed of a monomeric, enzymatically active A subunit which is responsible for ADP-ribosylation of GTP-binding proteins, and a non-toxic B subunit which binds receptors on the surface of the target cell and delivers the A subunit across the cell membrane.
In the case of CT and LT, the A subunit is known to increase intracellular cAMP levels in target cells, while the B subunit is pentameric and binds to GM1 ganglioside receptors. (LT-B also binds additional receptors.)
Previously, observations were made demonstrating that CT is able to induce mucosal and systemic immunity against itself when administered intraduodenally (i.e. to a mucosal surface). The membrane-binding function of CT was shown to be essential for mucosal immunogenicity, but cholera toxoid, also known as the B subunit of CT (CT-B) was inactive in isolation (Pierce and Gowans, J. Exp. Med. 1975; 142: 1550; Pierce, J. Exp Med. 1978; 148: 195–206).
Subsequently, it was demonstrated that native CT induced immunity to co-administered antigens (Elson, Curr. Top. Microbiol. Immunol., 1989; 146:29; Elson and Ealding, J. Immunol. 1984; 133:2892–2897; Elson and Ealding, Ibid. 1984; 132:2736–2741; Elson et al., J. Immunol. Meth. 1984; 67:101–118; Lycke and Homgren, Immunology 1986; 59:301–339) and that immune responses may be elicted to diptheria or tetanus toxoids when these antigens are applied to skin in combination with CT.
Two approaches have been taken in order to address the problem of CT toxicity. The first approach has involved the use of CT-B as a mucosal adjuvant. CT-B is entirely non-toxic. An adjuvant effect for co-administered CT-B has been alleged in a number of publications (Tamura et al., J. Immunol. 1992; 149:981–988; Hirabayashi et al., Immunology 1992; 75: 493–498; Gizurarson et al., Vaccine 1991; 9:825–832; Kikuta et al., Vaccine 1970; 8:595–599; Hirabayashi et al. Ibid. 1990; 8:243–248; Tamura et al., Ibid. 1989; 7:314–32-; Tamura et al., Ibid. 1989; 7:257–262; Tamura et al., Ibid. 1988; 6:409–413; Hirabayashi et al., Immunology 1991; 72:329–335 Tamura et al., Vaccine 1989; 7:503–505).
However, a number of aspects of the observations reported above were not entirely convincing. For example, it was noted that the adjuvant effect ascribed to CT-B was not H-2 (MHC) restricted. It is known, however, that the immune response to CTB is H-2 (MHC) restricted (Elson and Ealding, Eur. J. Immuno. 1987; 17:425–428). Moreover, the alleged adjuvant effect was observed even in individuals already immune to CT-B.
Other groups were unable to observe any mucosal adjuvant effect attributable to CT-B (Lycke and Holmgren, Immunology 1986; 59:301–308; Lycke et al., Eur. J. Immunol. 1992: 22:2277–2281). Experiments with recombinant CT-B (Holmgren et al., Vaccine 1993; 11:1179–1183) confirmed that the alleged effect is largely, if not exclusively, attributable to low levels of contamination of CT-B preparations with CT.
A second approach to eliminating the toxicity of CT has been to mutate the active holotoxin in order to reduce or eliminate its toxicity. The toxicity of CT resides in the A subunit and a number of mutants to CT and its homologue, LT, comprising point mutations in the A subunit, are known in the art. See, for example, International Patent Application W092/19265. It is accepted in the art that CT and LT are generally interchangeable, showing considerable homology. ADP-ribosylating bacterial toxin mutants have been shown to act as mucosal adjuvants, see International Patent Application W095/17211.